The alkalinizing, lysosomotropic agent ML-9 induces a pH-dependent depletion of ER Ca²⁺ stores in cellulo

ML-9 elicits a broad spectrum of effects in cells, including inhibition of myosin light chain kinase, inhibition of store-operated Ca²⁺ entry and lysosomotropic actions that result in prostate cancer cell death. Moreover, the compound also affects endoplasmic reticulum (ER) Ca²⁺ homeostasis, although the underlying mechanisms remain unclear. We found that ML-9 provokes a rapid mobilization of Ca²⁺ from ER independently of IP₃Rs or TMBIM6/Bax Inhibitor-1, two ER Ca²⁺-leak channels. Moreover, in unidirectional ⁴⁵Ca²⁺ fluxes in permeabilized cells, ML-9 was able to reduce ER Ca²⁺-store content. Although the ER Ca²⁺ store content was decreased, ML-9 did not directly inhibit SERCA's ATPase activity in vitro using microsomal preparations. Consistent with its chemical properties as a cell-permeable weak alkalinizing agent (calculated pKa of 8.04), ML-9 provoked a rapid increase in cytosolic pH preceding the Ca²⁺ efflux from the ER. Pre-treatment with the weak acid 3NPA blunted the ML-9-evoked increase in intracellular pH and subsequent ML-9-induced Ca²⁺ mobilization from the ER. This experiment underpins a causal link between ML-9's impact on the pH and Ca²⁺ dynamics. Overall, our work indicates that the lysosomotropic drug ML-9 may not only impact lysosomal compartments but also have severe impacts on ER Ca²⁺ handling in cellulo.